ROCK kinase was first characterized in 1996 by Matsui et al. as a Rho regulated kinase from the AGC subfamily of Ser/Thr kinases. [Matsui T, et al. EMBO J. 1996 May 1; 15(9):2208-16; Pearce L R, et al. Nat Rev Mol Cell Biol. 2010 January; 11(1):9-22; Olson M F, Curr Opin Cell Biol. 2008 April; 20(2):242-8]. Two genes encode the highly related enzymes ROCK1 and ROCK2, which are both widely distributed in many tissues [Mueller B K, Nat Rev Drug Discov. 2005 May; 4(5):387-98. Both contain a N-terminal kinase domain, a coil-coiled domain associated with dimerization, a mid-protein Rho-binding domain (RBD) and C-terminal, membrane association Plextrin homology (PH) domain, and a further C-terminal cysteine rich domain that binds lipid substrates [Pearce L R et al., Nat Rev Mol Cell Biol. 2010 January; 11(1):9-22; Mueller B K et al, Nat Rev Drug Discov. 2005 May; 4(5):387-98]. Binding of Rho-GTP to the Rho-binding domain releases auto-inhibition and allows the kinase to phosphorylate substrates. At least 11 different substrates have been demonstrated for ROCK kinases many of which are involved in the regulation of myosin-actin skeleton, and cell adhesion and mobility [Pearce L R et al., Nat Rev Mol Cell Biol. 2010 January; 11(1):9-22; Haas M A et al., J Neurosci Res. 2007 January; 85(1):34-46; Matsui T et al., J Cell Biol. 1998 Feb. 9; 140(3):647-57; Ivetic A et al., Immunology, 2004 June; 112(2):165-76]. The best characterized substrates are myosin light chain (MLC), and myosin phosphatase-1 (MYPT1) which together activate myosin and increase myosin fiber contraction; these actions lead to the tonic smooth muscle contraction in the vasculature, and lungs [Uehata M, et al., Nature 1997 Oct. 30; 389(6654):990-4; Kaneko-Kawano T et al., PLoS One 2012; 7(6):e39269].
The first synthetic kinase inhibitor to be approved was a ROCK inhibitor, Fasudil approved in 1995 for cerebral venospasm secondary to aneurysm, (Ono-Saito N, et al., Pharmacol Ther. 1999 May-June; 82(2-3):123-31). This compound was discovered while examining calmodulin inhibitors of the naphthalene-sulfonamide family, and noting a group that inhibited AGC family kinases; subsequently, ROCK was identified as the highest affinity target (Takayasu M, et al., J Neurosurg. 1986 July; 65(1):80-5; Ono-Saito N et al., Pharmacol Ther. 1999 May-June; 82(2-3):123-31; Uehata M et al., Nature 1997 Oct. 30; 389(6654):990-4). Other clinical studies have also shown that Fasudil leads to beneficial outcomes in angina (Shimokawa H et al., J Cardiovasc Pharmacol. 2002 November; 40(5):751-61), and pulmonary hypertension (Fukumoto Y et al., Heart 2005 March; 91(3):391-2; Fukumoto Y et al., Tohoku J Exp Med. 2007 April; 211(4):309-20). Trials for use of ROCK kinase inhibitors in glaucoma have been initiated with five different compounds (Chen J et al., Clin Ophthalmol. 2011; 5:667-77); however, early studies have shown that adequate pressure lowering and hyperemia—redness—might limit application (Williams R D, et al., Am J Ophthalmol. 2011 November; 152(5):834-41; Tanihara H, et al., Arch Ophthalmol. 2008 March; 126(3):309-15).
Human tissue and animal studies have shown that ROCK inhibitors have a role in wound healing (Bond J E et al., Plast Reconstr Surg. 2011 November; 128(5):438e-450e); treatment of spinal cord injury (Impellizzeri D et al., J Pharmacol Exp Ther. 2012 October; 343(1):21-33); and treatment of neuropathic pain (reviewed in Mueller B K et al., Nat Rev Drug Discov. 2005 May; 4(5):387-98). Human-genome-wide association studies connected the ROCK pathway with memory defects and Alzheimer's disease (Huentelman M J et al., Behav Neurosci. 2009 February; 123(1):218-23) which led to successful tests showing ROCK inhibitors improve memory in aged rats. Cardiac hypertrophy, and fibrosis in mice and rats are reduced by ROCK inhibitors, by a mechanism that may involve the ROCK mediated phosphorylation of PTEN-phosphatase and subsequent activation of the Akt pathway (reviewed in McKinsey T A et al., Nat Rev Drug Discov. 2007 August; 6(8):617-35). Together these findings indicate that ROCK inhibitors may find application in several important therapeutic areas.
Inflammation is strongly affected by ROCK inhibitors since cytoskeletal reorganization is important for the formation of the immune synapse and for chemotaxis (Takesono A. et al., PLoS One 2010 Jan. 19; 5(1):e8774; Hogg N et al., J Cell Sci. 2003 Dec. 1; 116(Pt 23):4695-705) Indeed, the beneficial effects of Fasudil on cerebral venospasm are associated with a reduction in the number of neutrophils at lesion sites (Satoh S et al., Jpn J Pharmacol. 1999 May; 80(1):41-8.). Inflammation suppression maybe part of the mechanism behind the beneficial effects of ROCK inhibitors on spinal cord injury (Impellizzeri D et al., J Pharmacol Exp Ther. 2012 October; 343(1):21-33) and arthritis (He Y et al., Arthritis Rheum. 2008 November; 58(11):3366-76). Given the effects on smooth muscle contraction and inflammation, which are the outcomes and the cause of asthma, respectively, asthma is an obvious application for ROCK inhibitors. In mice, guinea pigs and in human tissues, inhaled and systemic administration of ROCK inhibitors block eosinophilia, airway contraction and hyper-responsiveness, endpoints associated with asthma suppression. (Schaafsma D. et al., Respir Res. 2006 Sep. 26; 7:121; Kume H. et al., Curr Med Chem. 2008; 15(27):2876-85; Henry P J et al., Pulm Pharmacol Ther. 2005; 18(1):67-74; Hashimoto K et al., Thorax. 2002 June; 57(6):524-7; Schaafsma D et al., Eur J Pharmacol. 2006 Feb. 15; 531(1-3):145 50; Schaafsma D et al., Am J Physiol Lung Cell Mol Physiol. 2008 July; 295(1):L214-9; Schaafsma D et al., Eur J Pharmacol. 2008 May 13; 585(2-3):398-406; Iizuka K et al., Eur J Pharmacol. 2000 Oct. 13; 406(2):273-9; Yoshii A, Am J Respir Cell Mol Biol. 1999 June; 20(6):1190-200)
Compounds which can inhibit the biological moieties described above, or treat diseases involving those biological moieties, would be a significant advance in the art.